Abstract

The Generalized Phase Contrast (GPC) method of optical 3D manipulation has previously been used for controlled spatial manipulation of live biological specimen in real-time. These biological experiments were carried out over a time-span of several hours while an operator intermittently optimized the optical system. Here we present GPC-based optical micromanipulation in a microfluidic system where trapping experiments are computer-automated and thereby capable of running with only limited supervision. The system is able to dynamically detect living yeast cells using a computer-interfaced CCD camera, and respond to this by instantly creating traps at positions of the spotted cells streaming at flow velocities that would be difficult for a human operator to handle. With the added ability to control flow rates, experiments were also carried out to confirm the theoretically predicted axially dependent lateral stiffness of GPC-based optical traps.

Figures (6)

Schematic diagram of the experimental setup. The long working distance between the objective lenses significantly eases the insertion of a microfluidic system. The computer undertakes multiple tasks such as receiving feedback from an observation module, processing the acquired data and lastly generating control signals used for addressing the spatial light modulation module.

Left: Illustrating the particle dynamics in a lift and escape experiment. Forward movement is temporarily stopped while the optical trap lifts the particle. Right: Sequence of images showing yeast cells in a flow, 200 ms between successive frames. The yellow line represents the position of the trapped cell, which exits the trap between frame 3 and 4, the dark blue line indicates a free flowing cell. The exit velocity of the optically lifted yeast cell is greater than 60 µm/s, approximately 6 times that of a free flowing cell.

Mean values of escape velocities of 6 µm polystyrene beads as a function of pump rate. Particle size 5.68 µm. 50–100 data points per pump rate. Error bars are the std. deviation; the main contribution is pulsation of the syringe pump. Microbead escape velocity is significantly larger than measured for the yeast cells, due to the smaller size and higher refractive index of the polystyrene beads compared to that of the cell.

Realtime interactive manipulation of yeast cells in a microfluidic system (0.75 s between frames). Free moving cells are out of focus and creeping from left to right along the lower surface at ~10 µm/s. Five yeast cells are trapped (rightmost trap contains two cells). The yeast cells that are lifted into focus enter a region with flow velocity exceeding 50 µm/s (estimated by turning off traps). Frames 1–3: the lower cell is lifted. Frames 4–10: the upper and lower cells are repositioned by the user via computer mouse control.

(AVI: 2.5 MB) Detection and trapping of cells (0.75 s between successive frames). The square marks the detection/trapping area. When the square is off, the cells are released. The flow is set to 20 µl/hour, giving a cell velocity of approximately 15 µm/s. Note the two cells top-left in the detection area of frame 3; they are close together and both relatively small and therefore detected as one cell, resulting in the creation of a single common trap.